Fluorescent hplc assay for 20-hete and other p-450 metabolites of arachidonic acid

ABSTRACT

The present invention provides a fluorescent HPLC assay for detecting the presence and/or measuring the level of 20-hydroxyeicosatetraenoic acid (20-HETE) and other P-450 metabolites of arachidonic acid in a sample. P-450 metabolites of arachidonic acid are first extracted from the sample and then labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. The labeling reaction is catalyzed by N,N-diisopropylethylamine. Next, the labeled P-450 metabolites are separated on a 4.5×250-mm, 5 μM particle size C18 reverse-phase HPLC column using a mobile phase of methanol:water:acetic acid (82:18:0.1, v/v/v) and an isocratic elution at a rate of about 1.3 ml per minute. Fluorescence intensities of the column eluent are monitored by a fluorescence detector. Quantitation of P-450 metabolites in a sample can be made by using an internal standard.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application,Serial Number 60/196,076, filed on Apr. 10, 2000, which is incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agency: NIH Grant No: HL 29587, HL-36279, GM-31278, andHL-59996. The United States has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not applicable.

BACKGROUND OF THE INVENTION

A variety of diseases such as salt sensitive hypertension, toxemia ofpregnancy, asthma, hepatorenal syndrome, diabetes and subarachnoidhemorrhage are associated with abnormalities in arachidonic acid (“AA”)metabolism. Recent studies have indicated that AA is primarilymetabolized in the brain, kidney, lung, and vasculature by cytochromeP-450 enzymes to epoxyeicosatrienoic acids (EETs),dihydroxyeicosatrienoic acids (diHETEs), and 19- and20-hydroxyeicosatetraenoic acids (19- and 20-HETE). McGiff J C andQuilley J, Am J Physiol Regulatory Integrative Comp Physiol 277:R607-R623 (1999); Roman R J and Alonso-Galicia M, News Physiol Sci 14:238-242 (1999). 20-HETE and EETs are biologically active and have beenimplicated as paracrine factors and/or second messengers in theregulation of vascular tone, sodium and water excretion in the kidney,and airway resistance. McGiff J C and Quilley J, Am J Physiol RegulatoryIntegrative Comp Physiol 277: R607-R623 (1999); Roman R J andAlonso-Galicia M, News Physiol Sci 14: 238-242 (1999). Despite theimportance of P-450 metabolites of AA, very little is known about theregulation of the concentrations of these mediators in tissue andbiological fluids. Part of the problem has been the lack of a sensitive,inexpensive, and high-throughput assay to measure the endogenousconcentration of these compounds. To date, gas chromatography-massspectroscopy (GC-MS) with selective ion monitoring and one report of afluorescent enzyme based immunoassay (“EIA”) for EETs have been the onlymethods available to measure the concentration of P-450 metabolites ofAA in biological samples. Capdevila J H et al. J Biol Chem 267:21720-21726(1992); Catella F et al., Proc Natl Acad Sci USA 87:5893-5897 (1990); Prakash C et al., Biochem Biophys Res Commun 185:728-733 (1992); Schwartzman M L et al., Biochem Biophys Res Commun 180:445-449 (1991); Toto R et al., Biochem Biophys Acta 191: 132-134 (1987).The EIA requires a specific antibody that is no longer generallyavailable and, therefore, the assay cannot be reproduced in other labs.GC-MS has been successfully used to measure 20-HETE and EETs in theurine of humans and rats, and the reported concentration of thesemediators is in the range of 0.5-5 ng/ml. The urinary excretion of EETshas been reported to increase in rats fed a high-salt diet and inpatients with toxemia of pregnancy. Capdevila J H et al., J Biol Chem267: 21720-21726 (1992); Oyekan A O et al., J Clin Invest104:1131-1137(1999); Catella F et al., Proc Natl Acad Sci USA 87:5893-5897 (1990). Moreover, the urinary excretion of 20-HETE is elevatedin patients with hepatorenal syndrome and in DOCA-salt hypertensiverats. Sacerdoti D et al., J Clin Invest 100: 1264-1270 (1997); Oyekan AO et al., Am J Physiol Regulatory Integrative Comp Physiol 176:R766-R775 (1999).

Although GC-MS is a reliable method for the measurement of P-450metabolites of AA, the high cost for the purchase and maintenance of theinstrumentation and difficulties in preparing the samples for analysishave limited the use of this technique. Indeed, the preparation of urinesamples for GC-MS involves an organic extraction of the lipid fraction,separation of the EETs or HETEs fractions by thin-layer chromatographyand reverse-phase HPLC, derivatization of the samples to the methyl orpentabenzylfluoro esters, and conversion of these esters totrimethylsilyl derivatives. Toto R et al., Biochem Biophys Acta 191:132-134 (1987). It also requires the synthesis and addition of adeuterated internal standard to the samples to correct for variableextraction and derivatization efficiencies. The extensive samplepreparation reduces sample recoveries and the detection limits of thistechnique to the nanogram range. Oyekan A O et al., J Clin Invest 104:1131-1137 (1999); Schwartzman M L et al., Biochem Biophys Res Commun180: 445-449 (1991); Toto R et al. Biochem Biophys Acta 191: 132-134(1987). GC-MS is also limited to the measurement of a single compound ata time.

In the last few years, many fluorescent HPLC-based methods have beendescribed for the analysis of fatty acids following derivatization ofthe carboxyl or hydroxyl groups with agents such as anthryldiazomethane(ADAM), pyrenyldiazomethane (PDAM), bromomethyl- anddiethylaminocoumarin, 2-(2,3-naphthalimino)ethyltrifluoromethanesulfonate and other dyes. Brekke O L et al., J Lipid Res38: 1913-1922 (1997); Amet Y et al., J Chromatog B Biomed Appl 68:233-239 (1996); Minkler P E et al., Anal Biochem 231: 315-322 (1995);Yasaka Y and Tanaka M, J Chromatog B Biomed Appl 659:139-155 (1994);Yasaka Y et al., J Chromatog 508:133 (1990). Some of these studies havereported detection limits <10 pg for prostaglandins and fatty acids. Themain problems associated with these methods have been difficulties inobtaining consistent derivatization for lack of good catalysts, theinability to clearly resolve all of the P450 metabolites of AA found inbiologic samples by HPLC after these compounds have been reacted with afluorescent compound, and the lack of an internal standard with anextraction and labeling efficiency identical to the compounds ofinterest.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a fluorescent HPLC assay forsimultaneously detecting the presence and/or measuring the level of20-hydroxyeicosatetraenoic acid (20-HETE) and other P-450 metabolites ofAA in a sample. P-450 metabolites of AA are first extracted from thesample and then labeled with 2-(2,3-naphthalimino)ethyltrifluoromethanesulfonate, a fluorescent material. The labeling reactionis catalyzed by N,N-diisopropylethylamine. Next, the labeled P-450metabolites are separated on a 4.5×250-mm, 5 μM particle size C18reverse-phase HPLC column using a mobile phase of methanol:water:aceticacid (82:18:0.1, v/v/v) and an isocratic elution at a rate of about 1.3ml per minute. Fluorescence intensities of the column eluent aremonitored by a fluorescence detector.

When quantitation of P-450 metabolites in a sample is desired, a knownamount of an internal standard is added to the sample before theextraction of P-450 metabolites from the sample. The amount of a P-450metabolite in the sample can be calculated from the ratio of the P-450metabolite peak to the internal standard peak. When the HPLC assay ofthe present invention is used for detecting the presence of a P-450metabolite in a sample, the use of an internal standard is optional.

The HPLC assay of the present invention can be used for clinical testsof urine, blood, plasma, cerebrospinal fluid, bronchiolar lavage fluidand tissues for the diagnosis of diseases associated with abnormalitiesin the formation and/or levels of P450 metabolites of AA such as saltsensitive hypertension, toxemia of pregnancy, asthma, hepatorenalsyndrome, diabetes and subarachnoid hemorrhage.

It is an object of the present invention to provide a method tosimultaneously analyze more P-450 metabolites of AA in a sample thanprior art methods.

It is a feature of the present invention that the catalyst for thelabeling reaction allows the labeling of P-450 metabolites with a highdegree of consistency.

It is an advantage of the present invention that the method has a highdegree of intra-assay reproducibility.

It is another advantage of the present invention that the method has ahigh degree of quantitation sensitivity.

It is yet another advantage of the present invention that the method isrelatively simple and inexpensive to perform.

Further objects, features and advantages of the invention will beapparent from the following detailed description when taken inconjunction with the accompanying claims and drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a reverse-phase HPLC chromatogram demonstrating coelutionof fluorescent and radioactive peaks following derivatization of¹⁴C-labeled 20-hydroxyeicosatetraenoic acid [20-HETE; 10,000 counts/min(cpm)] with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate.

FIG. 2 shows representative HPLC chromatograms illustrating theseparation of fluorescently labeled 20-HETE from other P-450 metabolitesof arachidonic acid in a mixture of standards (A) and in a labeledsample (0.3 ml) of rat urine (B).

FIG. 3 is a liquid chromatography-gas chromatography chromatogram of the41-min HPLC peak corresponding to the retention time of fluorescentlylabeled 20-HETE.

FIG. 4 shows standard curves relating the ratio of areas of peaks to20-HETE and internal standards, either 19-hy-droxynonadeca-5(Z),8(Z),11(Z), 14(Z)-tetraenoic acid (19-OH) or WIT-002.

FIG. 5 shows effects of treatment of rat urine samples withglucuronidase on the concentration of 20-HETE.

FIG. 6 shows effects of blockade of the renal formation of 20-HETE with1-aminobenzotriazole (ABT; 50 mg/kg) on the urinary excretion of 20-HETEin spontaneously hypertensive rats (SHR) and Sprague-Dawley rats (SD).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for simultaneously analyzing20-HETE and other P-450 metabolites of AA using HPLC. By “analyzing,” wemean either detecting the presence and/or measuring the level of P-450metabolites of AA. As shown in the examples below, the HPLC assayresolved not only 20-HETE from all other known P-450 metabolites of AA,including 19-, 18-, 16-, 15-, 12-, and 5-HETE, 5,6-diHETE and EETs, butalso these other known P-450 metabolites of AA from each other with theexceptions of 17- and 16-HETE.

In one embodiment of the present invention, P-450 metabolites of AA froma biological sample are analyzed. By “biological sample,” we mean afluid or tissue from an animal or a human being, cultured cells ormicroorganisms, and medium or substrate used to culture the cells ormicroorganisms. Examples of a fluid from an animal or human beinginclude but are not limited to urine, blood, plasma, cerebrospinal fluidand bronchiolar lavage fluid. Examples of a tissue from an animal orhuman being include but are not limited to renal tissue, brain tissue,lung tissue, liver tissue, breast tissue and biopsies from suspectedcancerous tumors. In analyzing a biological sample, the sample is firstextracted for lipids and the lipids are then labeled with a fluorescentmaterial. Next the labeled lipid extract is loaded onto a HPLC columnfor separation of labeled P-450 metabolites of AA, which is followed bydetection of labeled P-450 metabolites of AA.

Methods of extracting lipids from a biological sample are known in theart. One method that can be used is described in detail below. It isappreciated that other lipid extraction methods can also be used.Preferably, as described below, the lipid extract is further purifiedbefore fluorescence labeling.

To fluorescently label the lipids, a fluorescent material and the lipidsare reacted with each other under the help of a catalyst. Many prior artfluorescent materials can label the lipids. For example, 9-ADAM, 1-ADAM,PDAM, 7-diethylaminocoumarin, and 4-bromomethyl-6,7-dimethoxycoumarincan be used to derivatize the samples using previously publishedtechniques. Nimura N and Kinoshita T, Anal Lett 13: 191-202 (1980);Metori A et al, J Chromatog 622: 147-151 (1993); Brekke O L et al., JLipid Res 38: 1913-1922 (1997); Balestrieri C et al., Anal Biochem. 233:145-150 (1996); Amet Y et al. J Chromatog B Biomed Appl 68:233-239(1996). However, when labeled with these materials, 20-HETEcannot be resolved from other HETEs (15-, 12-, 5-) and 5,6-diHETEs usingnormal phase, C18, or C8 reverse-phase HPLC. When resolving 20-HETE fromother HETEs (15-, 12-, 5-) and 5,6-diHETEs is not critical for aparticular assay, the above labeling material can be used.

As described below, a fluorescence label material that allows theresolution of the above closely related HETE structures is2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. Any method thatcan label a lipid sample with 2-(2,3-naphthalimino)ethyltrifluoromethanesulfonate may be used in the present invention. Forexample, 20-HETE and other P-450 metabolites may be labeled with2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate according to themethod of Yasaka et al. using potassium fluoride and 18-crown-6 ortetraethylammonium carbonate as a catalyst. Yasaka Y et al., J Chromatog508: 133 (1990). This technique works well with standards dissolved inorganic solvents. However, the labeling of biological samples is veryinconsistent because the reaction is inhibited by moisture. Thus, it ispreferable to use a catalyst that efficiently labels fatty acids even inthe presence of moisture. The present invention provides thatN,N-diisopropylethylamine is such a catalyst, which allows consistentlabeling of fatty acids by 2-(2,3-naphthalimino)ethyltrifluoromethanesulfonate.

After fluorescence labeling reaction is completed, it is preferable topurify the labeled lipids, e.g., to remove the non-reacted dyes, beforeloading the lipids onto a HPLC column.

The HPLC column used in the present invention is a 4.5×250-mm, 5 μMparticle size C18 reverse-phase HPLC column. The mobile phase used toelute the P-450 metabolites of AA from the column ismethanol:water:acetic acid with a volume ratio of about 82:18:0.1. Themobile phase passes through the column isocratically at a rate of about1.3 ml per minute.

The fluorescence intensity of 20-HETE and other P-450 metabolites of AAeluted from the column is detected by a fluorescence detector.

When the HPLC method of the present invention is used to quantitate theamount of 20-HETE and other P-450 metabolites of AA, a known amount ofan internal standard is added to the sample being analyzed. If thesample is a biological sample, an internal standard is added to thesample before the sample is extracted for lipids. Preferably, theinternal standard used has a similar extraction and labeling efficiencyas 20-HETE so that the amount of 20-BETE loaded on the column can bedetermined directly by comparing the ratio of the 20-BETE peak and theinternal standard peak. As described below, WIT-002 and19-hydroxynonadeca-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid are suitableinternal standards that have similar extraction and labelingefficiencies. However, a compound that is otherwise suitable as aninternal standard such as 10-hydroxyhexadecanoic acid tridecanoic acid,16-hydroxyhexadecanoic acid tridecanoic acid, 15-hydroxypentadecanoicacid and tridecanoic acid may also be used in the present invention evenif they have different extraction and labeling efficiencies from20-HETE. One of ordinary skill in the art knows however to correct forsuch differences. The calculation of the peak ratios and the amount ofdifferent P-450 metabolites of AA can be done manually or, preferably,aided by a computer.

The examples below show that 20-HETE quantitation sensitivity of theHPLC system of the present invention can reach as low as 1 to 10 ng whenthe fluorescence detector is set at a medium sensitivity (10×). Thisdegree of sensitivity is more than enough for routine measurements of20-HETE and other P-450 metabolites of AA in urine and renal tissuebecause the basal levels are about 10-100 ng/ml. However, when higherquantitation sensitivity is required, the fluorescence detector can beset at the high gain setting (100×) so that the sensitivity can reach1-100 pg.

The method for simultaneously analyzing 20-HETE and other P-450metabolites of AA provided by the present invention can be used forclinically diagnosing of a disease associated with abnormalities in theformation and/or levels of P450 metabolites of AA such as salt sensitivehypertension, toxemia of pregnancy, asthma, hepatorenal syndrome,diabetes and subarachnoid hemorrhage.

EXAMPLES

Methods

1. Reagents.

All chemicals used were of analytic or IPLC grade. The fluorescentprobes, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate, 9-ADAM,7-diethylaminocoumarin, 1-ADAM, 4-bromomethyl-6,7-dimethoxycoumarin, and3-PDAM were all purchased from Molecular Probes (Eugene, Oreg.). Thecatalyst N,N-diisopropylethylamine and 1-aminobenzotriazole (ABT) werepurchased from Sigma Chemical (St. Louis, Mo.). 5,6-diHETE, 5,6-EET, andthe 15-, 12-, and 5-HETE standards were from Biomol (Plymouth Meeting,Pa.). An internal standard,19-hydroxynonadeca-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid, and 8,9-,11,12-, and 14,15-diHETE, 16-, 18-, 19-, and 20-HETE, and 8,9-, 11,12-and 14,15-EET were synthesized by J. R. Falck (Univ. of TexasSouthwestern Medical Center, Dallas, Tex.). Another internal standard,20-5(Z),14(Z)-hydroxyeicosadienoic acid (WIT-002) was synthesized andkindly provided by Taisho Pharmaceutical (Saitama, Japan). HPLC-grademethanol was purchased from VWR (South Plainfield, N.J.), and aceticacid was purchased from Fisher Scientific (Pittsburgh, Pa.).

2. Lipid Extraction.

An internal standard (100 ng/ml) of a nonbiologically relevant hydroxyfatty acid such as WIT-002,19-hydroxynonadeca-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid,10-hydroxyhexadecanoic acid, 16-hydroxyhexadecanoic acid, or15-hydroxypentadecanoic acid was added to the samples. Alonso-Galicia Met al., Physiol Renal Physiol 277: F790-F796 (1999). The acidic lipidswere extracted with 3 vols of ethyl acetate after 0.25 ml of urine, 100μg of renal tissue homogenate, or 100 μl of buffer collected from amicrodialysis probe was acidified to pH 3.0 with formic acid. Thesamples were dried down under argon, reconstituted in 0.5 ml of 20%acetonitrile:water (pH 3.0), and applied to a Sep-Pak Vac 1 cc (catalogno. WAT054955; Waters, Milford, Mass.) that was prewashed with 1 ml ofwater followed by 1 ml of acetonitrile and 1 ml of water. The column waswashed twice with 1 ml of 30% acetonitrile:water to remove polar lipidsand then eluted with 400 μl of 90% acetonitrile:water. The eluent wasdiluted with 900 μl of water and applied to a prewashed Sep-Pak Vaccolumn and then eluted with 500 μl of ethyl acetate to capture the freefatty acids. The sample was taken to dryness under argon.

3. Fluorescent Labeling and HPLC Analysis of Lipids.

In preliminary experiments, standards and samples were derivatized with9-ADAM, 1-ADAM, PDAM, 7-diethylaminocoumarin, and4-bromomethyl-6,7-dimethoxycoumarin using previously publishedtechniques. Nimura N and Kinoshita T, Anal Lett 13: 191-202 (1980);Metori A et al., J Chromatog 622: 147-151 (1993); Brekke O L et al., JLipid Res 38: 1913-1922 (1997); Balestrieri C et al., Anal Biochem. 233:145-150 (1996); Amet Y et al., J Chromatog B Biomed Appl 68:233-239(1996). Although we were able to completely derivatize thesamples, we could not find a solvent system that would resolve labeled20-HETE from other HETEs (15-, 12-, 5-) and 5,6-diHETEs using normalphase, C18, or C8 reverse-phase HPLC. The only label that we found thatwas capable of resolving these closely related structures was2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. We initiallyattempted to label 20-HETE and other P-450 metabolites according to themethod of Yasaka et al. with 2-(2,3-naphthalimino)ethyltrifluoromethanesulfonate using potassium fluoride and 18-crown-6 ortetraethylanunonium carbonate as a catalyst. Yasaka Y et al., JChromatog 508: 133 (1990). Although this technique works well withstandards dissolved in organic solvents, the labeling of biologicsamples is very inconsistent because the reaction is inhibited bymoisture. We searched for another catalyst and found thatN,N-diisopropylethylamine catalyzes this reaction much moreconsistently.

To label the fatty acids, we resuspended samples, which were extractedand dried under argon, in 20 μl of acetonitrile containing 36.4 mM2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate.N,N-diisopropylethylamine (10 μl) was added to catalyze the reaction.The sample was reacted for 30 min at room temperature. The reactionswere dried down under argon, resuspended in 1 ml of 20%acetonitrile:water, and applied to a Sep-Pak Vac column. The column waswashed with 6 ml of 50% acetonitrile:water solution to remove unreacteddye and eluted with 500 μl of ethyl acetate. The eluent was dried underargon, and the samples were resuspended in 100 μl of the IPLC mobilephase (methanol:water:acetic acid, 82:18:0.1 vol/vol/vol). A 20-μlaliquot of the derivatized sample was separated on a 4.6×250-mmSym-metry C18 reverse-phase HPLC column (Waters, Milford, Mass.)isocratically, at a rate of 1.3 ml/min using methanol:water:acetic acidat 82:18:0.1 vol/vol/vol as a mobile phase. We tested various other C18reverse-phase HPLC columns and found that the Symmetry column was theonly one that could resolve all of the labeled P-450 metabolites of AAfrom each other. Fluorescence intensity was continuously monitored usinga fluorescence detector (model L-7480; Hitachi, Naperville, Ill.) at amedium gain sensitivity. The amount of 20-HETE in the sample wasdetermined by comparing the area of the 20-HETE peak to that of aninternal standard. Between samples, the column was flushed for 10 minwith a solution containing methanol plus 50% tetrahydrofuran to removeany residual labeled fatty acids.

4. GC-MS Confirmation of Labeled 20-HETE Peak.

20-HETE (10 ng) was added to a 1-ml sample and derivatized with2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. The fractioncontaining the fluorescent peak (retention time 41 min) was collectedand dried under argon. The derivatized sample was redissolved in 49.95%methanol, 49.95% water, and 0.1% formic acid and introduced into anelectrospray mass spectroscopy source at a rate of 5 μl/min using aHarvard syringe pump (Harvard Apparatus, South Natick, Mass.). Massspectral data were acquired over a mass-to-charge ratio range of200-1,200 at the rate of 5 s/scan with a Quattro II triple quadropolemass spectrometer (Micromass, Manchester, UK) fitted with anelectrospray source.

5. Biological Validation of the 20-HETE Assay.

Experiments were performed on 10- to 12-wk-old male Sprague-Dawley ratsand spontaneously hypertensive rats (SHR) purchased from Harlan SpragueDawley (Indianapolis, Ind.). The rats were housed in pairs in adedicated animal facility with a 12:12-h light-dark cycle and allowedfree access to a standard rat chow and drinking water. All procedureswere approved by the Animal Care and Use Committee at the MedicalCollege of Wisconsin.

6. Effects of an Irreversible P-450 Inhibitor on the Urinary Excretionof 20-HETE.

SHR and normotensive Sprague-Dawley rats were placed in specialmetabolic cages (model 650-00350; Nalgene, Rochester, N.Y.) thatefficiently separated urine from food and feces and were allowed toequilibrate for 5 days. After 5 days, the food was withdrawn from therat to prevent contamination of the urine sample and a 24-h controlurine sample was collected on dry ice. After the control sample wascollected, food was returned to the rats, and after a 2-day recoveryperiod, the rats received an intraperitoneal injection of ABT (50 mg/kg)followed by a second (50 mg/kg) injection 12 h later. Two hours afteradministration of the first dose of ABT, food was withdrawn and a 24-hexperimental urine sample was collected on dry ice. After the urine wascollected, the rats were killed with pentobarbital sodium (100 mg/kgip), and the kidneys were collected. The renal cortex was separated fromthe renal medulla, frozen in liquid nitrogen, and stored overnight formeasurement of the renal metabolism of AA. Briefly, the renal cortex washomogenized in a 10 mM potassium phosphate buffer (pH 7.7) containing250 mM sucrose, 1 mM EDTA, and 10 mM magnesium chloride, and microsomeswere prepared by differential centrifugation. P-4504A enzyme activitywas assayed by incubating renal cortical microsomes (0.5 mg) for 15 minat 37° C. with [1-¹⁴C]AA (0.1 μCi, 42 mM; Amersham, Arlington Heights,Ill.) in 1 ml of a 0.1 M potassium phosphate buffer (pH 7.4) containing1 mM NADPH as previously described. Ma Y H et al., Am J PhysiolRegulatory Integrative Comp Physiol 267: R579-R589 (1994). The reactionswere terminated by acidification to pH 4 using 0.1 M formic acid andextracted with ethyl acetate. Metabolites were separated using a25-cm×2-mm inner diameter (Supelco, Bellefonte, Pa.) C18 reverse-phaseHPLC column and a linear elution gradient ranging fromacetonitrile:water:acetic acid (50:50:0.1 vol/vol/vol) toacetonitrile:acetic acid (100:0.1 vol/vol) over a 40-min period. Theradioactive products were monitored using a radioactive flow detector(model 120; Radiomatic Instrument, Tampa, Fla.).

7. Measurement of 20-HETE Levels in Renal Interstitial Fluid ofAnesthetized Rats.

Male Sprague-Dawley rats were anesthetized with ketamine (30 mg/kg im)and Inactin (50 mg/kg ip). The femoral artery and vein were cannulatedfor measurement of blood pressure and intravenous infusions, and theureters were cannulated for collection of urine. The animal received anintravenous infusion of 0.9% NaCl containing 1% albumin at a rate of 6ml/h during the experiment. A microdialysis probe (BioanalyticalSystems, West Lafayette, Ind.) was implanted 3 mm deep into the renalcortex of the left kidney and perfused with sterile saline at a rate of10 μl/min. The animal was allowed to equilibrate for 1 h, and urine andmicrodialysis fluid were collected on ice for 1 h.

8. Statistics.

Data presented are means ±SE; n is the number of samples measured. Thesignificance of differences in mean values was analyzed using a pairedor unpaired t-test. A p<0.05 was considered to be statisticallysignificant.

Results

1. Assessment of the Efficiency of the Extraction Procedure and LabelingReaction.

Experiments were performed to determine the recovery of ¹⁴C-labeled20-HETE following extraction from urine with ethyl acetate and partialpurification using a Sep-Pak Vac column. Mean recovery of labeled20-HETE averaged 95±3% (n=6).

The effects of dye concentration and reaction time on the efficiency ofthe fluorescent derivatization reaction were also evaluated. ¹⁴C-labeled20-HETE was derivatized with 2-(2,3-naphthalimino)ethyltrifluoromethanesulfonate, and the fraction of fluorescently labeled andunreacted 20-HETE was determined by HPLC using a fluorescence detector(model L-7480, Hitachi) and a radioactivity detector (model 120;Radiomatic Instrument, Tampa, Fla.) arranged in series. The fluorescentand radioactive peaks coeluted (FIG. 1), indicating that the fluorescentpeak was derivatized ¹⁴C-labeled 20-HETE. The absence of a radioactivepeak at 10 min, which corresponds to the retention time of unreacted¹⁴C-labeled 20-HETE, indicates that the fluorescent labeling reactionwas complete under the present experimental conditions. In otherexperiments, we determined that the labeling reaction did not reachcompletion, and two radiolabeled peaks were seen when the concentrationof dye in the reaction was reduced or the reaction time was shortened to10 min.

2. Separation of Fluorescently Labeled 20-HETE.

A typical HPLC chromatogram illustrating the separation of the 20-HETEpeak from other structurally similar P-450 metabolites of AA ispresented in FIG. 2A. Abbreviations used in FIG. 2 include: EET,epoxyeicosatrienoic acid; diHETE, hydroxyeicosatetraenoic acid; WIT-002,20- hydroxyeicosa-6(Z),15(Z)-dienoic acid. As shown in FIG. 2A,fluorescently labeled 20-HETE elutes with a retention time of 41 min andis clearly separated from the peaks corresponding to labeled 14,15- and8,9-diHETE, 15-, 12-, and 5-HETE, and 8,9-EET. A more complete listingof the retention times of all the other biologically relevant P-450metabolites of AA and other potential interfering endogenous fatty acidsthat we tested is presented in Table 1. Even some of the compounds thatare quite difficult to resolve from unlabeled 20-HETE, such as 19-, 18-,and 16-HETE and 5,6- and 8,9-di-HETE, can be easily resolved fromfluorescently labeled 20-HETE using this HPLC system. The followingabbreviations are used in Table 1: DiHETE, dihydroxyeicosatetraenoicacid; ETYA, 5,8,11,14-eicosatetraynoic acid; HETE,hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; WIT-002,20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; and C19 analog,19-hydroxynonadeca-5(Z),8(Z),11(Z),14(Z)-tetranoic acid.

A typical chromatogram of a derivatized sample of urine collected from aconscious rat is presented in FIG. 2B. This sample contains peaks for11,12-EET and 8,9-diHETE and 20-HETE. It also contains peakscorresponding to the retention times of the subterminal HETEs (18-, 17-,16-, 15-, 12-, and 5-HETE), and EETs. The peak seen at 80 mincorresponds to the retention time of the internal standard, WIT-002.There is a small peak that appeared at 70 min when blank samplescontaining only dye and catalyst were injected. It therefore representseither a contaminant in the reagents or a fluorescent product formedfrom the dye and the catalyst. Unfortunately, this contaminant coeluteswith 11,12-EET, an important P-450 metabolite of AA. However, themagnitude of this blank peak is usually small (<150 mV) and can besubtracted from sample chromatograms by the computer.

Other experiments were performed to confirm that the peak that elutes at41 min was labeled 20-HETE. In these experiments, 20-HETE (100 ng/ml)was added to a blank sample, extracted, and derivatized, and the 41-minpeak was collected and analyzed using liquid chromatography (LC)-GC-MS.The results of these experiments, presented in FIG. 3, confirm that thispeak consists of a compound with mass-to-charge ratios (m/z) of 544,566, and 584, corresponding to the expected mass-to-charge ratios offluorescently labeled 20-HETE, 20-HETE plus a sodium ion, and 20-HETEplus a potassium ion, respectively.

TABLE 1 Retention times of major P-450 metabolites of arachidonic acidand 20- HETE analogs. P-450 Metabolites Retention Times, min14,15-diHETE 22 11,12-diHETE 25 C19 analog 28 15-Hydroxypentadecanoic 30acid 8,9-diHETE 30 ETYA 35 5,6-diHETE 35 19-HETE 39 20-HETE 4116-Hydroxyhexadecanoic acid 42 18-HETE 43 17-HETE 45 16-HETE 45 15-HETE46 10-Hydroxyhexadecanoic acid 47 12-HETE 49 5-HETE 62 Dimethyl 20-HETE65 14,15-EET 68 11,12-EET 70 8,9-EET 78 PS C19 analog 79 WIT-002 815,6-EET 90

3. 20-HETE Assay.

Standard curves were generated, in which samples containing variousamounts of 20-HETE (5-200 ng/ml) and 100 ng/ml of an internal standard,either WIT-002 or 19-hydroxynonadeca-5(Z),8(Z),11(Z),14(Z)-tetraenoicacid, were extracted and fluorescently labeled with2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. Aliquots of thesesamples containing 1-10 ng of 20-HETE and 5 ng of the internal standardwere separated by reverse-phase HPLC using an isocratic elution with 82%methanol:water at a rate of 1.3 ml/h. The ratio of the areas of the20-HETE and internal standard peaks were plotted against the amount of20-HETE in the aliquot and are presented in FIG. 4. The ratio of peakareas was highly correlated to the expected amount of 20-HETE in thesample (r²=0.98). Correlation coefficients averaged 0.99 for the curverelating 20-HETE and 19-OH (n=3 samples) and 0.99 for the curve relating20-HETE and WIT-002 (n=3 samples). The slopes of the curves were 0.19for WIT-002 and 0.26 for19-hydroxynonadeca-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid. Thesecoefficients are within errors associated with the addition of equalnanogram amounts of standards to samples and, for all practicalpurposes, indicate that 20-HETE and these two closely related analogslabel with equal efficiency. This is not the case with all compounds.For example, we found that other fatty acids that do not have a doublebond near the carboxyl group, such as linolenic and y-linolenic acids,and other unsaturated fatty alcohols, such as 10-hydroxyhexadecanoicacid, 16-hydroxyhexadecanoic acid, or 15-hydroxypentadecanoic acid,label with a much higher efficiency (typically 3:1) than 20-HETE, HETEs,and EETs. They are also not extracted from biological samples with thesame efficiency as 20-HETE.

Previous studies have indicated that the majority of 20-HETE and otherRETEs in human urine is excreted as a glucuronide. Prakash C et al.,Biochem Biophys Res Commun 185: 728-733 (1992). We therefore examinedthe effects of treatment of rat urine samples with glucuronidase (50units/ml) at 37° C. for 1 h on the concentration of 20-HETE. The resultsare presented in FIG. 5. Values are means ±SE; n=no. of samplesmeasured. There was no significant difference in the 20-HETE levelmeasured before and after treatment of the samples with glucuronidase.

We also measured 20-HETE levels after adding known amounts of thiscompound to urine samples and determined the intra-assay variation ofthe assay. Mean recovery of 20-HETE added to samples of rat urine was95±3% (n=6). Intra-assay variation in repeated measurements of the samesample of urine made over a period of several days averaged 4.7±1%(n=6).

4. Effect of a P-450 Inhibitor on the Urinary Excretion of 20-HETE.

The effect of ABT on urinary excretion of 20-HETE in SHR andSprague-Dawley rats is presented in FIG. 6. Values are means ±SE; n=no.of samples measured; *,§ indicate significant difference from controlwithin a group. Control urinary excretion of 20-HETE was significantlyhigher in SHR (n=4) than in Sprague-Dawley rats (n=5) and averaged1,334±337 and 872±338 ng/day, respectively. The excretion of 20-HETEfell by 65±10% after administration of ABT to SHR and by 74±9% inSprague-Dawley rats. The change in the urinary excretion of 20-HETEparalleled the fall in the renal production of 20-HETE in microsomesprepared from the kidneys of these animals. In this regard, 20-HETEproduction by renal cortical microsomes averaged 459±192 and 12±4pmolmin⁻¹mg⁻¹ in vehicle- and ABT-treated SHR (n=4), respectively.Similarly, 20-HETE production fell from 324±25 to 25±3 pmolmin⁻¹mg⁻¹in microsomes prepared from the kidneys of vehicle- and ABT- treatedSprague-Dawley rats (n=5).

5. Measurement of 20-HETE Levels in Renal Interstitial Fluid and RenalCortical Tissue.

Experiments were also performed to determine whether the assay wassensitive enough to measure the levels of 20-HETE and other P-450metabolites of AA in 100 μl of fluid collected from a microdialysiscapsule acutely implanted in the renal cortex and brain of rats andperfused with sterile saline at 5 μl/min. 20-HETE, diHETEs, and EETscould be detected in samples collected from both the kidney and thebrain. In the kidney, basal 20-HETE concentration averaged 3.1 ±0.3ng/ml (n=4). This finding compared with a concentration of 20-HETE inrenal cortical tissue of 1.8±0.3 ng/g of tissue (n=3). In microdialysisfluid collected from the brain of rats, we found that the concentrationsof 20-HETE and diHETEs averaged 27±3 and 39±1 ng/ml, respectively (n=5).

We claim:
 1. A method for analyzing P-450 metabolites of arachidonicacid from a biological sample, comprising the steps of: extracting P-450metabolites of arachidonic acid from the sample; labeling P-450metabolites of arachidonic acid with a fluorescent material; loadingP-450 metabolites of arachidonic acid labeled with the fluorescentmaterial onto a 4.5×250-mm, 5 μM particle size C18 reverse-phase HPLCcolumn; passing a mobile phase of methanol:water:acetic acid with avolume ratio of about 82:18:0.1 through the column isocratically at arate of about 1.3 ml per minute; and monitoring fluorescence intensityof eluent.
 2. The method of claim 1, further comprising the step of:purifying P-450 metabolites of arachidonic acid after extracting themfrom the sample but before labeling them.
 3. The method of claim 1,further comprising the step of: purifying P-450 metabolites ofarachidonic acid labeled with the fluorescent material after labelingP-450 metabolites of arachidonic acid with the fluorescent material butbefore loading them onto the HPLC column.
 4. The method of claim 1,wherein the fluorescent material is 2-(2,3-naphthalimino)ethyltrifluoromethanesulfonate.
 5. The method of claim 1, wherein thelabeling is catalyzed by a catalyst.
 6. The method of claim 5, whereinthe catalyst is selected from the group consisting of a combination ofpotassium fluoride and 18-crown-6, a combination of potassium fluorideand tetraethylammonium carbonate, and N,N-diisopropylethylamine.
 7. Themethod of claim 5, wherein the catalyst is N,N-diisopropylethylamine. 8.The method of claim 1, wherein the biological sample is selected fromthe group consisting of urine, blood, plasma, cerebrospinal fluid,bronchiolar lavage fluid and a tissue.
 9. The method of claim 8, whereinthe tissue is selected from the group consisting of renal tissue, braintissue, lung tissue, liver tissue, breast tissue and biopsies fromsuspected cancerous tumors.
 10. A method for analyzing P-450 metabolitesof arachidonic acid in a biological sample, comprising the steps of:adding an internal standard into the sample; extracting P-450metabolites of arachidonic acid and the added internal standard from thesample; labeling P-450 metabolites of arachidonic acid and the internalstandard with a fluorescent material; loading P-450 metabolites ofarachidonic acid and the internal standard, both of which are labeledwith the fluorescent material, onto a 4.5×250-mm, 5 μM particle size C18reverse-phase HPLC column; passing a mobile phase ofmethanol:water:acetic acid with a volume ratio of about 82:18:0.1through the column isocratically at a rate of about 1.3 ml per minute;monitoring fluorescence intensity of eluent; and quantitating P-450metabolites of arachidonic acid by calculating the peak ratio of ametabolite to the internal standard.
 11. The method of claim 10, whereinthe internal standard is a nonbiologically relevant hydroxy fatty acid.12. The method of claim 11, wherein the nonbiologically relevant hydroxyfatty acid is selected from the group consisting of WIT-002,19-hydroxynonadeca-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid,10-hydroxyhexadecanoic acid tridecanoic acid, 16-hydroxyhexadecanoicacid tridecanoic acid, 15-hydroxypentadecanoic acid and tridecanoicacid.
 13. The method of claim 10, wherein the internal standard isselected from the group consisting of WIT-002 and19-hydroxynonadeca-5(Z),8(Z), 11(Z), 14(Z)-tetraenoic acid.
 14. Themethod of claim 10, wherein the quatitating step is aided by a computer.15. The method of claim 10, wherein the fluorescence material is2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate.
 16. The method ofclaim 10, wherein the labeling is catalyzed by a catalyst.
 17. Themethod of claim 16, wherein the catalyst is selected from the groupconsisting of a combination of potassium fluoride and 18-crown-6, acombination of potassium fluoride and tetraethylammonium carbonate, andN,N-diisopropylethylamine.
 18. The method of claim 16, wherein thecatalyst is N,N-diisopropylethylamine.
 19. The method of claim 10,wherein the biological sample is selected from the group consisting ofurine, blood, plasma, cerebrospinal fluid, bronchiolar lavage fluid anda tissue.
 20. The method of claim 19, wherein the tissue is selectedfrom the group consisting of renal tissue, brain tissue, lung tissue,liver tissue, breast tissue and biopsies from suspected canceroustumors.
 21. A method for clinically diagnosing of a disease associatedwith abnormalities in arachidonic acid metabolism, comprising the stepof: analyzing P-450 metabolites of arachidonic acid from a sample of ahuman being comprising the steps of: extracting P-450 metabolites ofarachidonic acid from the sample; labeling P-450 metabolites ofarachidonic acid with a fluorescent material; loading P-450 metabolitesof arachidonic acid labeled with the fluorescent material onto a4.5×250-mm, 5 μM particle size C18 reverse-phase HPLC column; passing amobile phase of methanol:water:acetic acid with a volume ratio of about82:18:0.1 through the column isocratically at a rate of about 1.3 ml perminute; and monitoring fluorescence intensity of eluent.
 22. The methodof claim 21, wherein the disease associated with abnormalities inarachidonic acid metabolism is selected from the group consisting ofsalt sensitive hypertension, toxemia of pregnancy, asthma, hepatorenalsyndrome, diabetes and subarachnoid hemorrhage.
 23. A method forclinically diagnosing of a disease associated with abnormalities inarachidonic acid metabolism, comprising the step of: analyzing P-450metabolites of arachidonic acid from a sample of a human beingcomprising the steps of: adding an internal standard into the sample;extracting P-450 metabolites of arachidonic acid and the added internalstandard from the sample; labeling P-450 metabolites of arachidonic acidand the internal standard with a fluorescent material; loading P-450metabolites of arachidonic acid and the internal standard, both of whichare labeled with the fluorescent material, onto a 4.5×250-mm, 5 μMparticle size C18 reverse-phase HPLC column; passing a mobile phase ofmethanol:water:acetic acid with a volume ratio of about 82:18:0.1through the column isocratically at a rate of about 1.3 ml per minute;monitoring fluorescence intensity of eluent; and quantitating P-450metabolites of arachidonic acid by calculating the peak ratio of ametabolite to the internal standard.
 24. The method of claim 23, whereinthe disease associated with abnormalities in arachidonic acid metabolismis selected from the group consisting of salt sensitive hypertension,toxemia of pregnancy, asthma, hepatorenal syndrome, diabetes andsubarachnoid hemorrhage.
 25. A method for labeling P-450 metabolites ofarachidonic acid, comprising the step of: mixing P-450 metabolites ofarachidonic acid, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonateand N,N-diisopropylethylamine.